Fungal infections constitute a major threat to an ever expanding spectrum of immune- and medically- compromised patients. The opportunistic pathogenic fungi Candida, Aspergillus and Cryptococcus spp. are among the most common etiologic agents of mycoses; but infections caused by other yeasts and moulds are on the rise as advances in modern medicine prolong lives resulting in increasingly susceptible and vulnerable patients. However, fungi have recently been referred to as the ?hidden killers/the neglected epidemic? due to a lack of concomitant public awareness in fungal disease. The limited arsenal of antifungal agents contributes to the unacceptably high morbidity and mortality rates associated with fungal infections. Fungi are eukaryotes, and there is a paucity of selective targets that can be exploited for antifungal drug development. As a consequence, the antifungal arsenal is exceedingly short, mostly limited to three classes: polyenes, azoles and echinocandins. However, high toxicity and the emergence of resistance are limited factors for their clinical usage. To make matters even worse, some of the most troublesome emerging fungal pathogens fall completely outside of the spectrum of activity of these current antifungals. Moreover, the antifungal pipeline in most pharmaceutical companies is essentially dry. To conquer this formidable challenge the current proposal uses a highly efficient approach consisting of screening a repurposing library to identify compounds with novel antifungal activity followed by advancing the development of the leading hit compounds by assessing their activity in murine models of fungal infections, with an overall focus on resistant infections. The main objectives for the R21 Phase are: i) to conduct a large-scale screening of Calibr?s ReFRAME chemical library to identify high value compounds with novel antifungal activity against Candida albicans biofilms and against multidrug resistant Candida auris, an emerging pathogen and increasing nosocomial threat, and ii) to confirm the antifungal activity and determine the antifungal spectrum of action of hits from primary screening. Upon achieving the set transitional milestones for selection of a limited number of promising leads, the specific aims for the R33 Phase will be: i) to characterize the in vivo activity of the leading compounds in a number of clinically-relevant animal models of fungal infection that are fully established within the laboratories, and ii) to further characterize the in vitro antifungal activity of the leading compounds by testing them against an expanded number of clinical isolates of fungal species of interest, as well as testing their in vitro activity in combination with current existing antifungals. Altogether the strong translational impetus associated with the proposed studies, together with the complementary expertise of the assembled team of investigators covering from basic to clinical aspects of Medically Mycology, should maximize the chances for success and allow for accelerated development of new antifungal drugs, for which there is an urgent and dire need.